Methylammonium as a Transport Analog for Ammonium in Tomato (Lycopersicon esculentum L.)

Methylammonium (CH3NH3+) has been widely used as an analog of ammonium (NH4+) for examining transport in bacteria and fungi. We compared the kinetics of root CH3NH3+ and NH4+ uptake from solution culture in intact tomato (Lycopersicon esculentum cv T5) plants. Efflux of NH4+ and CH3NH3+ was negligible. The apparent maximum rate of absorption (apparent Vmax) was similar for NH4+ and CH3NH3+, but the apparent affinity (apparent Km) was about 10-fold greater for NH4+ than for CH3NH3+. In characterizing the interaction between NH4+ and CH3NH3+ transport, we used [15N]NH4+ and [14C]CH3NH3+ as well as improved methods for analysis of nonisotopic CH3NH3+ and NH4+. CH3NH3+ acted as an inhibitor of NH4+ influx. Relatively low concentrations of NH4+ strongly inhibited CH3NH3+ influx. Treatments with 1 mM methionine sulfoximine that blocked NH4+ assimilation had little influence on NH4+ inhibition of CH3NH3+ influx. These results suggest that the two ions share a common transport system in tomato, but because this transport system has a much greater affinity for NH4+, CH3NH3+ may be used as a transport analog only when ambient concentrations of NH4+ are very low.     

Enzymic synthesis of sym-homospermidine in Lathyrus sativus (grass pea) seedlings.

An enzyme catalysing the synthesis of sym-homospermidine from putrescine and NAD+ with concomitant liberation of NH3 was purified 100-fold from Lathyrus sativus (grass pea) seedlings by affinity chromatography on Blue Sepharose. This thiol enzyme had an apparent mol.wt. of 75000 and exhibited Michelis-Menten kinetics with Km 3.0mM for putrescine. The same enzyme activity could also be demonstrated in the crude extracts of sandal (Santalum album) leaves, but with a specific activity 15-fold greater than that in L. sativus seedlings.     

Cyanamide: a new substrate for nitrogenase

(1) Cyanamide (N identical to C-NH2) has been shown to be a substrate for purified Mo-nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum, with apparent Km values near 0.8 mM. (2) Reduction products were CH4, CH3NH2 and NH3 formed by pathways requiring 6 or 8 electrons: N identical to CNH2 + 6e + 6H+----CH3NH2 + NH3; N identical to CNH2 + 8e + 8H+----CH4 + 2NH3 (3) Acetylene reduction and hydrogen evolution were inhibited more than 75% by cyanamide (10 mM). Cyanamide also inhibited total electron flux at nitrogenase protein component ratios (Fe/MoFe) near 10. (4) Cyanamide was also a substrate for the recently isolated Va-nitrogenase of A. chroococcum, but with an apparent Km of 2.6 mM showed weaker binding and an 8-fold lower Vmax than did either Mo-nitrogenase. (5) The component ratios of nitrogenase proteins favouring CH4 formation was 3.5 Fe/MoFe protein and 1 Fe/VaFe protein.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Some properties of the pyruvate carboxylase from Pseudomonas fluorescens.

The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold from cells grown on glucose at 20 degrees C. The activity of this purified enzyme was not affected by acetyl-coenzyme A or L-aspartate, but was strongly inhibited by ADP, which was competitive towards ATP. Pyruvate gave a broken double reciprocal plot, from which two apparent Km values could be determined, namely 0-08 and 0-21 mM, from the lower and the higher concentration ranges, respectively. The apparent Km for HCO3 at pH 6-9, in the presence of the manganese ATP ion (MnATP2-), was 3-1 mM. The enzyme reaction had an optimum pH value of 7-1 or 9-0 depending on the use of MnATP2- or MgATP2-, respectively, as substrate. Free Mg2+ was an activator at pH values below 9-0. The enzyme was strongly activated by monovalent cations; NH4+ and K+ were the better activators, with apparent Ka values of 0-7 and 1-6 mM, respectively. Partially purified enzymes from cells grown on glucose at 1 or 20 degrees C had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after pre-incubation for 10 min at 46 degrees C. The molecular weight was estimated to be about 300000 daltons by gel filtration on Sephadex G-200. The regulatory properties and molecular weight are thus similar to those determined for the pyruvate carboxylases from Pseudomonas citronellolis and Azotobacter vinelandii.     

Purification and various properties of pantothenate kinase from the rat liver

Homogeneous (according to disc gel electrophoresis data) ATP: D-pantothenate-4'-phosphotransferase (pantothenate kinase, EC 2.7.1.33) was obtained from rat liver cytosol of heterogeneous stock rats. The enzyme was purified 199-fold with a 9.3% yield. The enzyme was relatively unstable but retained its activity in the presence of 10% glycerol containing 5.10(-4) M ATP over 10 days at 4 degrees C. The pH optimum was 6.5; the apparent Km values were equal to 1.2 X 10(-5) M and 1.4 X 10(-3) M for pantothenate and ATP, respectively, at the ATP/Mg2+ ratio of 1. Pantetheine produced a competitive inhibition of pantothenate kinase. Pantethine or pantetheine disulfide did not inhibit the enzyme.     

Lactose and D-galactose metabolism in Staphylococcus aureus. III. Purification and properties of D-tagatose-6-phosphate kinase

D-Tagatose-6-phosphate kinase, an inducible enzyme that functions in the metabolism of lactose and D-galactose in Staphylococcus aureus, was purified about 300-fold from an extract of D-galactose-grown cells. The enzyme catalyzed the nucleoside triphosphate-dependent phosphorylation of both D-tagatose 6-phosphate and D-fructose 6-phosphate. Although the Vmax values were equal for these two substrates, the apparent Km values differed by 10,000-fold, being 16 micro M for D-tagatose 6-phosphate and 150 mM for D-fructose 6-phosphate. The purified enzyme was free from the constitutive D-fructose-6-phosphate kinase. Phosphoryl donors used by D-tagatose-6-phosphate kinse, listed in order of decreasing rates at saturating concentrations were GTP, UTP ITP ATP, CTP, and TTP; the Km values were 0.38, 0.91, 0.17, 0.16, 18, and 20 mM, respectively. The enzyme appeared to be nonallosteric; it exhibited Michaelis-Menten kinetics and was not inhibited by high concentrations of MgATP. However, it was activated 3- to 4-fold by 33.3 mM K+, NH4+, Rb+, and Cs+, and was inhibited 31 to 65% by 33.3 mM Na+ and Li+. It was inactivated reversibly by the thiol reagent, N-ethylmaleimide. The subunit molecular weight was estimated to be 52,000, and the native enzyme appeared to be a dimer with a sedimentation coefficient of 6.8 S. Data on stability, pH optimum, and inducibility of the enzyme are also presented.     

The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ.

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.     

Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax).

A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38 000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1-0.2 M citrate buffer at 30 degrees C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 muM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5% of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.     

Role of monovalent cations in reactions catalyzed by glycerol dehydrase from Aerobacter aerogenes]

Cobamide-dependent glyceroldehydrase (GDH) is shown to have an absolute requirement in monovalent cations: K+, NH4+, Tl+, Rb+ and Cs+. Dependencies of initial dehydratation rates of three substrates: glycerol, ethyleneglycol and 1,2-propandiol on the concentration of K+ are studied. Km values for K+, NH4+ and Tl+ are calculated to be 7-10-3, 4-10-3 and 1-10-3 M respectively. Effect of K+ on Km values for glycerol and coenzyme and on maximal reaction rate is investigated. It is shown that the apparent affinity of the substrate to the enzyme does not depend on monovalent cation; the apparent affinity of the coenzyme somewhat changes with the change of K+ concentration. Maximal reaction rate increases with the increase of K+ content. On the basis of kinetic data obtained possible mechanism of the activating effect of monovalent cations in reactions, catalyzed by GDH, is discussed.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Kinetics and regulation of the myofibrillar adenosine triphosphatase.

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+.     

Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii

Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii. It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S. The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group. pCMB inhibited the enzyme activity (IC50 = 2 microM). The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C. It is activated by 0.1 M KCl and KI and 2 M urea. 3-4 M urea completely inactivated the enzyme. Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.     

Investigation of sarcoplasmic reticulum SH-groups]

The amount and the reaction capacity of the thiol groups in the sarcoplasmic reticulum containing up to 86% of Ca-ATPase were determined using 7-chloro-4-nitrobenzo-2-hydroxo-1,3-diazole (NBD-chloride). The total amount of SH-groups interacting with NBD-chloride is about 9 moles/10(5) g of protein as determined in the excess of NBD-chloride (750 micrometers). With respect to their sensitivity to NBD-chloride the SH-groups may be divided into two classes: slow and fast ones (5,3 and 3,5 moles/10(5) g of protein, respectively). The modification constants for the fast and slow SH-groups are 0,16 and 0,015min-1. ATP (30 micrometers) decreases the number of fast groups by 1 mole/10(5) g of protein. At higher concentrations of ATP (1--3 mM) the amount of fast SH-groups is decreased by 3 moles/10(5) g of protein, their modification rate constant being decreased 2-fold. ATP at concentration of 1 mM, decreases the rate constant for the Ca-ATPase inactivation by NBD-chloride from 0.68 down to 0,073 min-1, which coincides with the modification rate constant for fast SH-groups (0,071 min-1) under the same conditions. Ca2+ at concentration of 10(-4) M increases the amount of fast thiol groups by 1 mole/10(5) g of protein, the rate constant of their modification by NBD-chloride being increased 2-fold. A half-maximal effect was observed in the presence of 5.10(-7) M Ca2+ . Mg2+ did not affect the total amount of fast thiol groups; however, it decreased their modification rate constant.     

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.     

The influence of basic residues on the substrate specificity of protein kinase C

The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.     

NAD+-kinase from Neurospora crassa: isolation and properties

The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively.     

Kinetic evaluation, using 13N, reveals two assimilatory nitrate transport systems in Klebsiella pneumoniae.

A kinetic evaluation of initial rates of nitrate transport at concentrations between 1 microM and 1 mM indicated the presence of two transport processes. Analysis of the contribution of each process to the total activity permitted the determination of kinetic constants (Km) of 4.9 microM and 4.2 mM for the high-and low-affinity systems, respectively. The ratio of the maximal velocity of the high-affinity system to that of an apparent low-affinity system was about 0.3. Both systems were inhibited by the presence of NH4+ in the transport assay. Growth in the presence of equimolar NO3- and NH4+ repressed the synthesis of both systems when compared with growth in NO3- alone.     

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.